The long-term objective is to explore the relationship between the ascending and descending auditory pathways at the level of the superior olive complex. Two general groups of olivary cells are the source of the descending pathways. One set of pathways projects mainly to the cochlea via the olivocochlear bundle (OCB). The second set of pathways uses other routes and only projects to the cochlear nucleus. These descending pathways have been implicated in such basic auditory functions as protecting cochlear hair cells from over-stimulation, facilitating frequency and interaural auditory discriminations and aiding speech comprehension in noisy environments. The applicants will focus on one specific and one general question, concerning olivary input to the cochlear nucleus and the source of ascending input to both groups of descending olivary cells. Do the olivary cells which project to the cochlear nucleus form a single generalized pathway or are there several parallel pathways projecting from the superior olive to the cochlear nucleus? One discrete pathway was recognized in the previous grant period and several others were suggested. The cells and axon projections of these pathways will be characterized by intracellular labeling, in a tissue slice preparation of the auditory brainstem. Prior to slice preparation, cell soma will be "pre-tagged" by the retrograde transport fluorescent dyes injected into the cochlear nucleus. Fluorescently pre-tagged cells are then targeted within the slice preparation for more complete axonal and dendritic labeling with intracellular electrodes. In corollary experiments, pre-tagged OCB cells will be intracellularly re-labeled and characterized by their dendritic and axon projections. How do the ascending auditory pathways to the superior olive contact the cells which form the descending pathways to the cochlea and cochlear nucleus? The connections formed by one ascending pathway were described as part of the original specific aims. These findings raise the possibility that some or all of the several distinct pathways which ascend from the cochlear nuclei to the superior olive may be matched with distinct subpopulations of descending olivary cells. Intra- and extracellular labeling techniques will be used to trace ascending axons which contact labeled descending cells within the superior olive. Fluorescent dyes are used for intracellular labeling, as filled cells and processes can be seen and directly traced within physiologically maintained tissue slices. To examine filled cells with electron microscopy, the applicants will use antibodies to two fluorescent intracellular dyes and more permanent, electron-dense secondary antibodies. In this way, fluorescently labeled synaptic and dendritic profiles can be recognized and analyzed at the ultrastructural level. The data gathered in these studies will provide a structural basis for understanding the functional role of olivary descending pathway in audition.